p53 andINK4a/ARF mutations promote tumorogenesis and drug
resistance, in part, by disabling apoptosis. We show that
primary murine lympomas also respond to chemotherapy by engaging
a senescence program controlled by p53 and p16INK4a. Hence,
tumors with p53 or INK4a/ARF mutations-but not those lacking ARF
alone - respond poorly to cyclophosphamide therapy in vivo.
Morover, tumors harboring a Bcl2-mediated apoptotic block
undergo a drug-induced cytostasis involving the accumulation of
p53, p16INK4a, and senescence markers, and typically acquire p53
or INK4a mutations upon progression to a terminal stage.
Finally, mice bearing tumors capable of drug-induced senescence
have a much better prognosis following chemotherapy than those
harboring tumors with senescence defects. Therefore, cellular
senescence contributes to treatment outcome in vivo.

Figure
1. Contribution of p53 and Bcl2 to Treatment Responses

Mice harboring ctrl.-MSCV, p53 null-MSCV, and ctrl.-bcl2
lymphomas were treated at comparable tumor burdens (day
0) with a single dose of cyclophosphamide (CTX) and
monitored by whole-body fluorescence imaging.
Representative examples are shown.

Whole body fluorescence imaging of
lymphoma progression in live mice. The cover shows the temporal
and spatial progression of Em-myc lymphoma cells tagged with
green fluorescent protein in a live mouse (with time progression
from top to bottom). Note that the lymphomas first expand within
the lympoid compartments and bone. In the absence of p53 or
following Bcl-2 overexpression, these lympomas readily
disseminate into nonlympoid compartments. For details see
Schmitt et al. (pp. 289-298) in this issue.

Summary

Although the p53 tumor supressor acts in plethora of
processes that influence cellular proliferation and survival, it
remains unclear which p53 functions are essential for tumor
suppression and, as a consequence, are selected against during
tumor development. Using a mouse model harbouring primary,
genetically modified myc-driven lympomas, we show that
disruption of apoptosis downstream of p53 by Bcl2 or a
dominant-negative caspase 9 confers-like p53 loss-a selective
advantage, and completely alleviates pressure to inactivate p53
during lymphomagenesis. Despite their p53-null-like aggressive
phenotype, apoptosis-defective lymphomas that retain intact p53
genes do not display the checkpoint defects and gross aneuploidy
that are charcteristic of p53 mutant tumors. Therefore,
apoptosis is the only p53 function selected against during
lymphoma development, whereas defective cell-cycle checkpoints
and aneuploidy are mere byproducts of p53 loss.

Lymphomas with indicated genotypes and transduced
with a GFP-coexpressing retrovirus were transplanted
into recipients to monitor lymphoma dissemination in
whole viable animals by GFP fluorescence.

REVIEW

Green Fluorescent Protein
Imaging of Tumor Cells in Mice

By Robert M. Hoffman

[LAB ANIMAL 31, No.
4, 34-41 April 2002]

ABSTRACT

The use of green fluorescent protein-expressing cancer cells
allow real-time, high-resolution imaging of tumor growth ,
metastasis, and angiogenesis, both in sity and
extrnally. The author reviews the use of GFP
technology for studying human cancer in mouse model.

The human pancreatic tumor cell line, BxPC-3,
was engineered to stably express high-levels of the Aquorea
victoria green fluorescent protein (GFP). The GFP-expressing
pancreatic tumor cell line was surgically orthotopically
implanted (SOI) as tissue fragments in the body of the pancreas
of nude mice. Intravital imaging was used for quantification of
growth metastasis on the liver. The diameters of the three
micrometastases were 288 mm and 208 mm the right lobe and 344 mm
on the left lobe as quantified by image analysis at day-70 post
SOI.

We report here whole-body optical imaging, in
real time, of genetically fluorescent pancreatic tumors growing
and metastasizing to multiple sites in live mice. The whole-body
optical imaging system is external and noninvasive. Human
pancreatic tumor cell lines, BxPC-3 and MiaPaCa-2, were
engineered to stably express high-levels of the Acqurea victoria
green fluorescent protein (GFP). The GFP-expressing pancreatic
tumor cell lines were surgically orthotopically implanted as
tissue fragments in the body of the pancreas of nude mice.
Whole-body optical images visualized real-time primary tumor
growth and formation of metastatic lesions that developed in the
spleen, bowel, portal lymph nodes, omentum, and liver.
Intravital imaging was used for quatification of growth of
micrometastasis on the liver and stomach. Whole-body imaging was
carried out with either a trans-illuminated epi-fluorescence
microscope or a fluorescent light box, both with a
thermoelectrically cooled color CCD camera. The simple,
noninvasive, and highly selective imaging made possible by the
strong GFP fluorescence allowed detailed simultaneous
quantitative imaging of tumor growth and multiple metastasis
formation of pancreatic cancer. The GFP imaging affords
unprecendented contionuous visual monitoring of malignant growth
and spread within intact animals without the need for anesthesia,
substrate injection, control agents, or restraint of animals
required by the other imaging methods. The GFP imaging
technology presented in this report will facilitate studies of
modulators of pancreatic cancer growth, including inhibition by
potential chemotherapeutic agents.

Visualization of GFP-Expressing
Tumors and Metastasis In Vivo

Robert M. Hoffman

[BIOTECHNIQUES 30,
No. 5 May, 2001]

ABSTRACT

We have developed mouse models of metastatic cancer with
genetically fluorescent tumors that can be imaged in fresh
tissue, in situ, as well as externally. To achieve this
capability, we have transduced the green fluorescent protein (GFP)
gene, cloned from the bioluminescent jellyfish Aequorea victoria,
into a series of human and rodent cancer cell lines that were
selected in vitro to stably express GFP in vivo after
transplantation to metastatic rodent models. Techniques were
also developed for transduction of tumors by GFP in vivo. With
this fluorescent tool, we detected and visualized for the first
time tumors and metastasis in fresh viable tissue or in situ in
host organs down to the single cell level. GFP tumors on the
colon, prostate, breast, brain, liver, lymph nodes, lung,
pancreas, bone and other organs can also be visualized
externally, transcutaneously by quantitative whole-body
fluorescence optical imaging. Real-time tumor and metastatic
growth and angiogenesis and inhibition by representative drugs
can be imaged and quantified for rapid anti-tumor,
anti-metastatic and anti-angiogenesis drug screening. The
GFP-transfected tumor cells enabled a fundamental advance in the
visualization of tumor growth and metastasis in real time in
vivo.